1. Field of the Invention
The present invention relates to methods for delivery of a biomolecule transduction complex (BTC), such as a fusion polypeptide, into cells. The biomolecule transduction complex (BTC) comprises a protein transduction domain (PTD) and a molecule of interest, such as a polypeptide.
2. Background Art
Generally, living cells are not permeable to macromolecules such as proteins and nucleic acids. The fact that only small-size molecules can permeate through the membrane of living cells at very low rates has restricted the research to develop drugs to cure, prevent, or diagnose diseases using macromolecules, such as, proteins and nucleic acids. Therefore, an effective method of transducing biologically active macromolecules into the cytosol and nucleus of living cells, without deleterious side effects, is needed.
Protein transduction domains (PTDs) have been used for delivery of biologically active molecules (Viehl C. T., et al., Ann. Surg. Oncol. 12:517-525 (2005); Noguchi H., et al., Nat. Med. 10:305-309 (2004); and Fu A. L., et al., Neurosci. Lett. 368:258-62 (2004)). However, no attempts have been made to use PTDs as a way of delivering a cytoplasmic domain of a receptor protein. Also, no attempts have been made for the intranasal delivery of a PTD with a biological molecule.
CTLA-4 (cytotoxic T lymphocyte antigen-4) is an activation-induced surface molecule on T cells and is essential for the negative regulation of T cell activation. It binds to B7-1 or -2 on antigen presenting cells (APC) with an affinity of 10- to 20-fold higher than that of CD28 which is a positive costimulatory molecule for T cell activation (Ngoc L. P., et al., Curr. Opin. Allergy Clin. Immunol. 5:161-166 (2005); Noel P. J., et al., Adv. Exp. Med. Biol. 406:209-217 (1996); and Perkins D., et al., J. Immunol. 156:4154-4159 (1996)).
The cytoplasmic domain of CTLA-4 containing Immunoreceptor Tyrosine-based Inhibitory Motif (ITIM) has been found to be 100% conserved among different species, suggesting that this domain is important for negative regulation of CTLA-4 on T cell activation by sequestering intracellular signaling molecules (Ravetch, J. V. & Lanier, L. L., Science 290:84-89 (2000); and Jay, C. U. & Jie, J., Curr. Opin. Immunol. 9:338-343 (1997)). Therefore, the cytoplasmic domain of CTLA-4 is an excellent molecular target for the development of immunotherapeutic drugs for asthma, autoimmune diseases and graft rejection. To date, attempts to deliver a receptor protein, in particular CTLA-4, into a cell using a PTD have been difficult. We have now shown a way to deliver CTLA-4 as a cargo for a PTD by fusing just the cytoplasmic domain of the receptor protein with a PTD. The cytoplasmic domain of CTLA-4 is specific to the activated T cells, so when used as a cargo protein for a PTD, it overcomes the lack of tissue specificity of the cationic PTDs which limited their in vivo utility.